Vitamins B.sub.12, folate, and B.sub.6 are required cofactors in metabolic pathways involving methionine, homocysteine, cystathionine, and cysteine. B.sub.12 in the form of 5'-deoxyadenosylcobalamin is an essential cofactor in the enzymatic conversion of methylmalonylCoA to succinylCoA. The remethylation of homocysteine (HC) to methionine catalyzed by methionine synthase requires folate (methyltetrahydrofolate) and B.sub.12 in the form of methylcobalamin. HC is condensed with serine to form cystathionine (CT) in a reaction catalyzed by cystathionine .beta.-synthase which requires B.sub.6 (pyridoxal phosphate). CT is hydrolyzed in another B.sub.6 -dependent reaction to cysteine and .alpha.-ketobutyrate.
It is important to diagnose and treat B.sub.12, folate, and B.sub.6 deficiencies because these deficiencies can lead to life-threatening hematologic abnormalities which are completely reversible by proper treatment. B.sub.12 deficiency is a multisystem disorder with extremely varied clinical presentation which has been thought to occur in 0.4% of the population, e.g., about 1 million people in the United States. Symptoms of B.sub.12 deficiency include significant anemia, displayed for example in decreased hematocrit (e.g., &lt;25%) or hemoglobin (e.g., .ltoreq.8 g%), with macrocytic red blood cells (i.e., mean cell volume generally greater than 100 fl), or neurologic symptoms of peripheral neuropathy and/or ataxia. See, for example, Babior and Bunn (1983) in Harrison's Principles of Internal Medicine, (Petersdorf et al., eds.), McGraw-Hill Book Co., New York; Lee and Gardner (1984) in Textbook of Family Practice. 3rd Ed. (Rakel, ed.), Saunders & Co., Philadelphia). The hematological abnormalities seen are due to intracellular folate deficiency since folate is required for a number of essential enzymatic reactions involved in DNA and RNA synthesis and since the form of folate in serum (5-methyltetrahydrofolate) must be metabolized to tetrahydrofolate by the B.sub.12 -dependent enzyme methionine synthase before it can be utilized by the RNA- and DNA-related enzymes. While it has been well recognized that individuals with B.sub.12 deficiency could display neurologic disorders in the absence of anemia, such situations were believed to be exceptional and rare. See, Beck (1985) in Cecil Textbook of Medicine, 17th Ed., (Wyngaarden and Smith, eds.), W. B. Saunders, Philadelphia, pp. 893-900; Babior and Bunn (1987) in Harrison's Principles of Internal Medicine, 11th Ed., (Braunwald et al., eds.) McGraw-Hill, New York, pp. 1498-1504; Walton (1985) in Brain's Diseases of the Nervous System, 9th Ed., Oxford University Press, Oxford, UK. The neurologic symptoms of B.sub.12 deficiency were considered to be late manifestations of the disease most typically occurring after the onset of anemia or, if they occurred first, were soon to be followed by the onset of anemia. See, Woltmann (1919) Am. J. Med. Sci. 157: 400-409; Victor and Lear (1956) Am. J. Med. 20: 896-911.
However, it has recently been shown that the textbook description of severe megaloblastic anemia and combined systems disease of the nervous system is the rarest presentation of B.sub.12 deficiency at the present time (Stabler et al. (1990) Blood 76: 871-881; Carmel (1988) Arch. Int. Med. 148: 1712-1714; Allen (1991) in Cecil Textbook of Medicine, 19th Ed., (Wyngaarden and Smith, et al. eds.), W. B. Saunders, Philadelphia, pp. 846-854.). Therefore, contrary to previous teachings, patients that may benefit from B.sub.12 therapy may have minimal to no hematologic changes while manifesting a wide variety of neurologic and psychiatric abnormalities (Lindenbaum et al. (1988) N. Engl. J. Med. 318: 1720-1728; Greenfield and O'Flynn (1933) Lancet 2: 62-63). This is particularly true for populations at risk for B.sub.12 deficiency, such as the elderly population (Pennypacker et al. (1992) J. Am. Geriatric Soc. 40: (in press).
The incidence of folate deficiency in the population is unknown, but has been thought to occur commonly in individuals with various degrees of alcoholism. The hematologic abnormalities seen with folate deficiency, such as macrocytic anemia, are indistinguishable from those seen with B.sub.12 deficiency. Folate is required for a number of essential enzymatic reactions involved in DNA and RNA synthesis, and is particularly important in rapidly dividing cells like those in the bone marrow.
B.sub.6 is required for the first step in heme synthesis and serves a major role in transamination reactions of amino acid metabolism, in decarboxylations, and in the synthesis of the neuroactive amines histamine, tyramine, serotonin, and .gamma.-aminobutyric acid (GABA). Clinical manifestations include microcytic hypochromic anemia, characteristic skin changes of dermatitis and acrodynia, muscular weakness, and a variety of neuropsychiatric abnormalities including hyperirritability, epileptiform confusions, depression and confusion (Newberne and Conner (1989) in Clinical Biochemistry of Domestic Animals, Academic Press, San Diego, pp. 796-834).
Vitamin deficiencies are generally determined by measurement of serum levels. Normal serum B.sub.12 levels are 200-900 pg/ml, with levels of less than 100 pg/ml being said to indicate clinically significant deficiency (Beck (1985) supra) However, serum B.sub.12 levels are a relatively insensitive determinant of B.sub.12 deficiency in that only 50% of patients with clinically confirmed B.sub.12 deficiency have levels less than 100 pg/ml, 40% are 100-200 pg/ml, and at least 5-10% have values in the 200-300 pg/ml range. Diagnosis is further complicated by the fact that 2.5% of normal subjects (6,250,000 people in the U.S.) have low serum B.sub.12 levels (Allen (1991) supra), with no evidence of B.sub.12 deficiency and are unlikely to benefit from B.sub.12 therapy (Schilling et al. (1983) Clin. Chem. 29: 582; Stabler (1990) supra).
Normal serum folate levels are 2.5-20 ng/ml, with levels less than 2.5 ng/ml indicating the possibility of clinically significant deficiency. Like B.sub.12 serum levels, however, serum folate levels are a relatively insensitive measure in that only 50-75% of patients with folate deficiency have levels less than 2.5% ng/ml, with most of the remaining 25-50% being in the 2.5-5.0 ng/ml range (Allen (1991) in Cecil Textbook of Medicine. 19th Ed., supra).
The development of sensitive serum metabolite assays for HC, CT, MMA, and 2-MCA has allowed the relationship between metabolite levels and vitamin deficiencies to be investigated (Stabler et al. (1987) Anal. Biochem. 162: 185-196; Stabler et al. (1986) J. Clin. Invest. 77: 1606-1612; Stabler et al. (1988) J. Clin. Invest. 81: 466-474). It has been found that elevated serum levels of HC and MMA are clinically useful tests of functional intracellular deficiencies of B.sub.12 and folate, with elevated HC levels seen with both B.sub.12 and folate deficiencies, and elevated MMA levels seen with a B.sub.12 deficiency (Allen et al. (1990) Am. J. Hematol. 34: 90-98; Lindenbaum et al. (1990) Am. J. Hematol. 34: 99-107; Lindenbaum et al. (1988) N. Engl. J. Med. 318: 1720-1728; Beck (1991) in Neuropsychiatric Consequences of Cobalamin Deficiency, Mosby Year Book 36: 33-56; Moelby et al. (1990) 228: 373-378; Ueland and Refsum (1989() J. Lab. Clin. Med. 114: 473-501; Pennypacker et al. (1992) supra). Increased serum levels of CT are seen in both deficiencies and 2-MCA is elevated in B.sub.12 deficiency (Allen et al. (1991) in Proceedings of the 1st International Congress on Vitamins and Biofactors in Life Science, Kobe (Japan); Allen et al. (1993) Metabolism (in press)). HC and CT may be elevated in patients with intracellular deficiency of B.sub.6, but this has not been as well documented (Park and Linkswiler (1970) J. Nutr. 100: 110-116; Smolin and Benvange (1982) J. Nutr. 112: 1264-1272).
Elevated serum metabolite levels are observed in disease states other than classic vitamin deficiencies. For example, elevated HC levels have been observed in the presence of vascular disease. The homocysteine theory of atherosclerosis, formulated by McCully and Wilson (1975) Atherosclerosis 22: 215-227, suggests that high levels of HC are responsible for the vascular lesions seen in homocystinuria, a genetic defect caused by a deficiency in the enzyme cystathionine .beta.-synthase. The theory also implies that moderate elevations of HC might be associated with increased risk for vascular disease (Ueland et al. (1992) in Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function (Francis, Jr., ed.), Marcel Dekker, Inc., New York, pp. 183-236). Moderate hyperhomocysteinaemia has been shown to be frequently present in cases of stroke and to be independent of other stroke risk factors (Brattstrom et al. (1992) Eur. J. Clin. Invest. 22: 214-221). Clinical and experimental evidence demonstrates that patients who are homozygotes for cystathionine .beta.-synthase deficiency have a markedly increased incidence of vascular disease and thrombosis. A number of studies (see, Clarke et al. (1991) N. Engl. J. Med. 324: 1149-1155) strongly suggest that heterozygotes for a deficiency of cystathionine .beta.-synthase also have an increased incidence of vascular disease and thrombosis and that such heterozygotes may constitute as many as one-third of all patients who develop strokes, heart attacks, or peripheral vascular disease under age 50. It is also likely that such heterozygotes are also at increased risk for vascular disease and thrombosis after age 50. Since the incidence of heterozygosity for cystathionine .beta.-synthase deficiency is estimated to be 1 in 60-70, this means that there are approximately 4 million heterozygotes in the U.S. It is also possible that patients with vascular disease due to other causes, such as hypercholesterolemia, would also benefit from a decrease in their serum HC levels even if their existing levels are only slightly elevated or actually within the normal range.
Renal disease is another condition that gives rise to elevated levels of serum metabolites. Approximately 75% of patients with renal disease have elevated serum concentrations of HC, CT, MMA, and 2-MCA. Since patients with renal disease have a significant incidence and marked acceleration of vascular disease, it might be beneficial to lower their serum metabolite levels, especially that of HC.
An increasing prevalence of low serum B.sub.12 concentrations with advancing age has been found by many but not all investigators (Bailey et al. (1980) J. Am. Geriatr. Soc. 28: 276-278; Eisborg et al. (1976) Acta Med. Scand. 200: 309-314; Niisson-Ehle et al. (1989) Dig. Dis. Sci. 34: 716-723; Norman (1985) 33: 374; Hitzhusen et al. (1986) Am. J. Clin. Pathol. 85: 3236), folate (Magnus et al. (1982) Scan. J. Haematol. 28: 360-366; Blundell et al. (1985) J. Clin. Pathol. 38: 1179-1184; Elwood et al. (1971) Br. J. Haematol. 21: 557-563; Garry et al. (1984) J. Am. Geriatr. Soc. 32: 71926; Hanger et al. (1991) J. Am. Geriatr. Soc. 39: 1155-1159), and B.sub.6 (Ranke et al. (1960) J. Gerontol. 15: 41-44; Rose et al. (1976) Am. J. Clin. Nutr. 29: 847-853; Baker et al. (1979) J. Am. Geriatr. Soc. 27: 444-450). Moreover, prevalence estimates for these vitamin deficiencies vary widely depending on the population groups studied. It has been unclear whether this increased prevalence is a normal age related phenomena or a true reflection of tissue vitamin deficiency and whether the low serum vitamin concentrations are a reliable indicator of functional intracellular deficiency.
It is difficult, expensive and time-consuming to distinguish between deficiencies of vitamins B.sub.12, folate, and B.sub.6. The hematologic abnormalities seen with B.sub.12 deficiency are indistinguishable from those seen with folate deficiency. Similarly to a B.sub.12 deficiency, B.sub.6 deficiencies also result in hematologic as well as neuropsychiatric abnormalities. The traditional methods of determining deficiencies by measurement of serum vitamin levels are often insensitive. As a result, in order to determine if and which vitamin deficiency is present, a patient will be treated with one vitamin at a time and the response to that vitamin determined by normalization of serum vitamin levels and the correction of hematologic abnormalities. These steps are then repeated with each vitamin. This method of treatment is both expensive and time-consuming. In the presence of multiple deficiencies, the diagnosis of vitamin deficiencies is further confused and give rise to the dangerous possibility that only one deficiency will be treated. For example, the hematologic abnormalities seen with a B.sub.12 deficiency will respond to treatment with folate alone. However, the neuropsychiatric abnormalities caused by the B.sub.12 deficiency will not be corrected and may indeed by worsened.
It has now been discovered for the first time that the prevalence of intracellular deficiencies of vitamins B.sub.12, folate, and B.sub.6, alone or in combination, is substantially higher than that previously estimated by measurement of serum vitamin concentrations. The present disclosure establishes that tissue deficiencies of one or more of the vitamins B.sub.12, folate and B.sub.6, as demonstrated by the elevated metabolite concentrations, occurs commonly in the elderly population even when serum vitamin levels are normal. Based on this new discovery, the present invention addresses the problem of distinguishing between vitamin deficiencies when low, low-normal, or normal serum vitamin concentrations are found by providing formulations for the treatment of high serum metabolites and at-risk populations for combinations of one or more tissue deficiencies of vitamins B.sub.12, folate, and B.sub.6.
Hathcock and Troendle (1991) JAMA 265: 96-97, have suggested the treatment of pernicious anemia with an oral pill containing 300 to 1000 ug or more per day of B.sub.12. However, contrary to the present invention, Hathcock and Troendle teach away from combining B.sub.12 therapy with folate, since "if the oral cobalamin therapy should fail to maintain adequate levels, folate might provide protection against development of anemia while permitting nerve damage from cobalamin deficiency."
U.S. Pat. No. 4,945,083, issued Jul. 31, 1990 to Jansen, entitled: Safe Oral Folic-Acid-Containing Vitamin Preparation, describes a oral vitamin preparation comprising 0.1-1.0 mg B.sub.12 and 0.1-1.0 mg folate for the treatment or prevention of megaloblastic anemia. This formulation presents a problem in the case of a B.sub.12 deficient patient, in that the 0.5 mg folate may correct the hematologic abnormalities present, but the 0.5 mg B.sub.12 dose may be insufficient to correct a B.sub.12 deficiency due to inadequate intrinsic factor. By contrast, the formulation of the present invention teaches the use of the combination of B.sub.12 and folate, and of B.sub.12, folate and B.sub.6, sufficient to treat either single or multiple deficiencies of B.sub.12, folate, and B.sub.6. The present invention does not rely on the determination of vitamin deficiencies by the measurement of serum vitamin levels, but uses the more sensitive measurement of elevated serum metabolites of HC, CT, MMA, and 2-MCA, shown to be related to the presence of B.sub.12 and/or folate and/or to B.sub.6 deficiencies or to the presence of the increased risk of neuropsychiatric, vascular, renal, and hematologic diseases.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.